JPH0269637A - Method and device for measuring concentration of chromophore substance - Google Patents

Abstract

PURPOSE: To reduce the amount of errors by minimizing the influence of noise by passing flash light through a sample and measuring generated absorbancy values at different wavelengths at the time of measuring the concentration of a chromophoric material existing in the sample and its variation by using a clinical chemical analyzer. CONSTITUTION: A beam 11 of pulsed light from a xenon flash lamp 10 is reflected by means of a toroidal mirror 12 and the reflected beam 13 of light is transformed into a divergent beam 17 of light through a cuvette 14, a lens 15, and a slit 16. Then the beam 17 is reflected into parallel beams 19 of light by a first collimator mirror 18 and the beams 19 are projected upon a diffraction grating 20. Reflected light 120 from the grating 20 is reflected into a plurality of convergent beams 22 of light having different wavelengths by a second collimator mirror 21 and the beams 22 are separately made indent to a detector row through the slit row 25 of an order classification filter. A computer program is prepared at the detector row 24 and transmitted to an electronic unit 26. The unit 26 decides the category which is the sum total of the concentration, main chromophore, and various errors and statistically displays the category.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to clinical chemistry analyzers, systems and methods. In particular, it relates to a device for measuring the concentration and changes in concentration of chromophoric substances that are indicative of the amount of a particular analyte present in a sample.

BACKGROUND OF THE INVENTION Different systems are known for measuring the concentration of chromophores in a sample. In one known form, these devices are based on a steady state light bulb source that passes a steady beam of light through the sample. The steady state bulb source uses a tungsten-halogen filament. The power output of such bulbs is about 50 to 100 watts, and steady state operation requires exposure to the bulb source for anywhere from 15 minutes to over an hour to provide the sample with an operational analysis system. .

Other unique known steady state systems use a first beam of light passing through the sample. A second beam is separated from the first beam and is used as a reference beam. The two rays are physically and spatially separated. Electronic switching means expose the detector and measurement electronics alternately, and once non-simultaneously, to measure multiple wavelengths coming from each beam of the stationary light source. This is complex, and switching back and forth between physically spaced beams does not provide the desired precision system.

In yet other known systems, - sets of optical elements are used in which - reference beams followed by - sampling beams pass alternately through the optical system. These two rays are separated in time. A sample is placed in the sample holder when the sampling beam is passed through. There is no sample in the holder when the reference beam is passed through. This approach is therefore relatively cumbersome due to the need to change the sample in the holder over time.

In another form of the device, a ray of light coming from the steady state source is such that a small portion of the ray passes through a reference detector, and
The beam is split such that more than 95% of the light beam passes through the sample and a detector associated with the sample. This system is
Examine the full spectrum of white light effects in determining the sample analysis.

All these known systems have in common that they use two beams. The measurement of light is the total white light spectrum or specific wavelengths.

The analysis to obtain the concentration determination of the analyte is performed using the formula Abs
= −log (1/I.) as determined from the measurement of the absorbance of the chromophore given by: (2) indicates the intensity measured through the sample, and Io indicates the intensity of the reference beam. These intensities are measurements of white light generated in the manner of two rays or specific wavelengths.

Absorbance is related to the concentration of chromophore, expressed in the formula Abs -Ebc. where E is the extinction coefficient for the absorption capacity element, b is the cell path length, and C is the concentration.

The specific requirements for known systems using a single beam are:
This means that extremely high stability of light source operation is required to obtain measurement results with the required precision. For really accurate measurements, it is necessary to have a high photostability of the order of about 1710% over 1 hour. Relatively expensive techniques and components must be used to achieve this stability. Because of this high degree of stability, relatively fast operating procedures and analysis systems are used. However, as noted, this is expensive due to the components and methodology required to obtain a high degree of photostability.

With increasing throughput demands for accurate analysis and measurement in the medical, chemical, and quantitative fields, it has become important to accurately provide high-speed measurements on systems containing large numbers of samples.

Current known systems have sufficient accuracy and
Does not provide fast enough throughput at an acceptable relatively low cost. There is a need here to provide a system that can quickly provide analysis of a large number of samples.

SUMMARY OF THE INVENTION In accordance with the present invention, a single beam system is contemplated. The instability of the light source to obtain the same accuracy criteria is higher than in conventional systems. That is, it exceeds about 1% over the course of a chemical reaction of about 15 minutes. This increased instability allows for increased throughput at lower cost. High efficiency results in accuracy are achieved by the inventive flash correction technique. Additionally, the speed at which the system operates, i.e. the interval between successive flashes, is limited by thousands of seconds, as opposed to two beam systems or known single beam systems, which require the bulb's output to last many seconds. It is one minute of a second.

In accordance with the present invention, a photometer for measuring changes in concentration in at least one chromophoric substance uses a flashbulb and multiple wavelength flash correction methodology. The amount of absorbance for at least two, and preferably up to five, selected wavelengths is measured for each flash, and the concentration of the chromophore is determined by an appropriate linear combination of the multiple absorbance values. This linear combination applies to the set of absorbances fixed coefficients k that are dependent on a set of extinction coefficients e and a matrix f of coefficients representing the noise pattern in the photometer.

An optimal linear combination that minimizes noise artifacts is established for at least two different selected wavelengths, thereby reducing the amount of error in concentration measurements. This optimal linear combination takes into account the nature of the correlation in the errors at different wavelengths, and also takes into account the direction in which the chemical reaction proceeds, ie determined by the chemi-extinction coefficient.

Because of the one-thousandth of a second flash rate, which gives higher intensity than steady-state light bulb sources, and the measurement means and flash correction techniques of the invention, the overall system of the invention is less sensitive from a stability standpoint, but the final Fast in terms of throughput. The intensity of a flashbulb is about 50,000 watts with a flash of about 1 to 5 microseconds. In the invention's analysis system, one
Throughput rates for a range of samples are obtained.

Therefore, instead of a steady white light bulb as a reference signal, the present invention is directed to a high intensity flash bulb operable during short pulse widths, ie, microseconds. Instead of effectively stabilizing the system with a complex set of components, with the stability obtained from using the best chosen linear combination of absorbance values for two or more wavelengths produced by the instantaneous beam, A single beam flash system operates. The linear combination of absorbance is equal to the logarithm of the product of the power of intensity values at different wavelengths. Optimal linear coupling effectively increases the signal effect and reduces the noise effect of the light source or flash bulb.

The method adopted in the present invention maintains high precision through the use of a flashbulb to generate the light beam, as opposed to a steady state white light source, while increasing speed and throughput. Furthermore, since the light bulb is not lit during that time, it will not burn out quickly. Costs are not increased due to mobile downtime for replacement and recalibration. It also generates less heat. These elements are also
Thus providing a simpler system with high throughput.

On the other hand, flash sources present the problem that the flashes are not always the same. Therefore, the amount and nature of light passing through the sample will vary from flash to flash. This problem, however, is eliminated by the present invention, which employs a strong correlation in intensity changes at different wavelengths in each flash.

Flashing white light bulbs have the property of varying intensity for each flash because the arc between the two electrodes generates variable energy. Additionally, the arc physically moves to a new position within the bulb, and this causes an image to change back and forth on a photodetector in a photoarray that receives the light after it has passed through the sample. . The optics associated with the arc also pass a portion of the different energies through a manual slit of a monochromator located downstream of the sample.

The present invention employs highly correlated noise associated with each flash at different wavelengths. Kneading reduces the effects of the noise and also provides a more accurate absorbance signal.

This provides more accurate concentration values. The signal to noise ratio is maximized by selecting an optimal linear combination of absorbance values at two or more wavelengths when estimating concentration.

Single-beam systems operate simultaneously with intensity measurements at different wavelengths. Compared to conventional devices, the need for intensity stabilization of the light source is therefore reduced.

Beyond the application of measuring absorbance and therefore changes in concentration in a single chromophore, this system
For example, it is sensitive to the presence of interfering substances such as lipids or hemoglobin in different samples. When such an interfering substance is present, the system adds information to the concentration result that the interfering substance adversely affects the concentration result.

The present invention also covers methods for determining the extinction coefficient for a chromophoric material, from absorbance measurements made with varying amounts of the chromophoric material to any increasing factor. This technique can also be used to demonstrate that a given chemical reaction has only one chromophore present as the reaction progresses.

For the photometer, it is possible to select a set of 5 different wavelengths from the available 10 wavelengths for measuring absorbance values for a given chemical reaction. - A set of five wavelengths often allows the most desirable use of absorbance values in obtaining estimates regarding the concentration of chromophores in chemical reactions.

If desired, output signals with more or less than five wavelengths can be utilized. In other cases, it is possible for a photometer monochromator to have more photoarray elements in the detector. The photometer is provided with a detector aperture and the photoarray is selected to be at one spaced apart location with respect to the different wavelengths.

Intensity signals at two or more wavelengths are obtained and an electronic unit processes the analog intensity values into digital absorbance values. A microprocessor program combines these absorbance values into a linear combination to obtain the chromophore concentration.

The monochromator system separates a flash of light passing through the sample into multiple wavelengths that are directed to a plurality of the detector apertures.

(Example) The Mikami photometer was equipped with a monochromator 20 along with a detector means.
4 and a flash bulb 10. The xenon flash bulb 10 is
It is arranged to send a white light beam 11 in the form of a pulse to a toroid mirror 12, which transmits a white light beam 1
3 to the sample holder or cuhet 14.

The cuvette 14 has a passage length for the light beam 13 of approximately 5 mm. After the light ray 13 passes through the cuvette 14,
The light rays directed toward the lens 15 and exiting from the lens 15 pass through the slit 16 as diverging light rays 17. This beam 17 is directed onto a first collimating mirror 18 . A parallel beam of light 19 is reflected from the first collimating mirror 18 and directed towards the diffraction grating 20 .

From a diffraction grating 20, each element of said beam is directed to a second collimating mirror 21.
It is spectrally or divided into 20 parts. The second collimator mirror 21 reflects the light beam 120 into a convergent light beam 22,
2 is directed towards a plane 23 displaying the distinct wavelengths of the light beam and on which a detector array 24 is arranged. In front of the detector row 24 there is an order sorting filter and slit row 25, which in total up to 10 detector rows 24 for distinct wavelengths.
allow passage to. The intensity signals of various wavelengths from the detector array 24 are conveyed to an electronic unit 26 for measurement and processing using a microprocessor or under control of a computer program.

Optical components are arranged inside the housing 27 and the entire photometer unit, including the electronic unit 26, is part of a chemical analyzer, which suitably places the sample at a location 28 within the housing 27. Includes means for automatically positioning and moving cuvettes 14. electronic unit 2
Suitable recording means are provided to record the data obtained from 6.

To determine the absorbance value and therefore the concentration value, five different wavelengths are selected from the ten useful wavelengths used in the process. These values are determined in such a way that the signal to noise ratio is maintained at a maximum.

Furthermore, the extinction coefficient of each chemical reaction is determined by the method described below.

These wavelengths are, for example, from 340 nm to 700 nm.
It is in the corpus of.

The value of absorbance measured during a −Δ reaction is determined by three influences: 1) the concentration of the main chromophore in a given reaction, whose concentration is determined;
It is the summation of 2) various possible interfering substances, and 3) various types of errors in absorbance measurements. In category 3), the influence of the type of flashbulb is important;
It is also this type and noise effect that is minimized.

Accurate estimates of the concentration of the main chromophore of a chemical reaction, as it changes during the reaction, are obtained despite the effects of said interferences and other types of errors.

From this concentration data, multiple endpoint values or multiple rate values are obtained. This in turn is used to determine the desired analyte concentration in the sample.

There are two types of category 2) interfering substances. First, there are multiple interfering substances introduced from the sample that remain unchanged during the reaction. These have an important relationship with end point chemical reactions. Rate chemistry uses the time error of absorbance changes that occur during the course of the reaction, and the presence of a constant offset from such interfering substances does not affect the rate value obtained. A second type of interfering substance participates in the reaction and varies in concentration with the concentration of the primary chromophore. This is important for both endpoint and rate chemistry.

Flash bulb changes play a significant role in these errors because they are relatively large. Changes in absorbance at various wavelengths are strongly correlated.

This correlation allows the estimation error to be substantially reduced by suitably adjusting the coefficients used in the concentration estimation of one chromophore.

The different extinction coefficients indicate the relative amount by which the absorbance changes at various selected different wavelengths. Multiple extinction coefficients are
This corresponds to the sampling of a special curve of the absorbance of the substance at various selected different wavelengths. Multiple extinction coefficients are
used to arrive at the best estimate for the concentration of the chromophore.

Errors in absorbance measurements can be characterized statistically by measuring the changes in absorbance that occur in deionized water over a large number of samples. A variance matrix F represents the statistical relationship resulting from a set of such measurements.

The estimate used for the concentration of the primary chromophore is a special linear combination of absorbance. Let the absorbance measured at n selected wavelengths be aI+ a2+...an,
kI aI, 2a2°·kTlaT+ is the estimated concentration value. Next, the coefficients are 2. ... The appropriate selection of kn is determined by the extinction coefficient of the chromophore and the dispersion matrix F for the selected set of wavelengths.

k, 2. . . . When obtaining k, it is assumed that no interfering substances exist. - only the chromophore and multiple absorbance measurement errors need to be considered. This method uses only the extinction coefficients of the chromophore and the dispersion matrix F of deionized water to obtain the single best solution that minimizes the mean squared error in the concentration estimate.

However, taking into account the possible presence of interfering substances, multiple linear equations are developed that determine the deviation of the measured absorbance from the direction of the assumed single chromophore extinction coefficient in various orthogonal directions. . There are limitations to each equation when taking into account absorbance measurement errors in each of these directions. If the limit is exceeded, this indicates that the interfering substance is present in excessive amounts and the concentration estimate is invalid.

Once the optimal estimate of chromophore concentration is reached, the absorbance is assumed to be due to a single chromophore and the error distributed according to the changes observed in the deionized water. This is expressed as Beer's law: a=c*e+f. where C is the scalar chromophore concentration, and a, e, and f are an nx1 matrix of measured absorbance values, the extinction coefficient of the chromophore, and the errors in absorbance, respectively.

Flashbulb 10 is used to measure changes in the concentration of a single chromophore substance, indicating the amount of a particular analyte present in a sample. This is done by measuring the amount of absorbance for up to five selected wavelengths in each flash. The chromophore concentration at each flash is then estimated by a suitable linear combination of multiple absorbance values.

In the absence of errors, multiple absorbance values at one or more wavelengths to measure the concentration of a single chromophore component constitute redundant information, so a large number of different linear combinations can be used equally well. Can be done. However, when a large amount of error exists, primarily caused by changes in the bulb 10 from flash to flash, an optimal linear combination is needed that minimizes the effect of these changes on the density determination.

Optimal linear combinations are found in using the correlation between multiple errors at different wavelengths, which reduces the amount of error present in the concentration estimate. The optimal linear combination reflects both the nature of the correlated errors and the "direction" in which the chemical action travels, ie, the extinction coefficient of the chemical action.

Assuming that it is an absorbance measurement or a suitable offset,
The average values of the errors f are zero. k is an l×n row matrix of coefficients of the desired optimal linear combination. The linear combination or matrix product c6
= ★a is the estimated value of C. The statistically estimated average value E of this estimated value is E (c6 ) = E (kAa) = c large (kAa) + large E (f) = ck (kAa).

To ensure that this estimated mean is the same as the actual value C, a constraint 'ke is placed on k. Therefore, the error in the estimated value can be expressed as ★f in Co −C=.

The second condition placed on k is that the mean squared error of this estimate is minimized. That is, E ((co c)2)”E ((k”f)2)=E
((k large f) large (k large f) ゛ )=to *E (f6f') 6 ゛ is the minimum, where the notation ° indicates the transposition of the matrix. The nxn dispersion matrix E(f6f') is denoted by F.

Therefore, given e and F, k is determined so that k6F'kk' is minimized, subject to k*e=1. The independent variable d of the differentiation at the minimum (k6F large')
=2dk*F*k' When using =0, always d(kAa)zdkbothe=0.

Therefore, F6' and e must be parallel. That is, for some scalar t, F'kk'
= *e or = t★e'6F-'. Here, F-1
is the inverse matrix of F. Next, t is determined by finding k★e=1. The final solution is = (e' *F - weight *6)
-'*6' *F-1.

The dispersion and covariance of f can be determined by observing the corresponding dispersion and covariance of absorbance occurring in a given medium in a cuvette, such as air, deionized water, or dye solution. The set of variances or covariances of these absorbances with respect to the average value of the absorbances for all possible bearings of wavelengths is the matrix F= for that set of wavelengths.
E (f large f') is constructed.

This calculation of F is done during the bulb calibration process and
This calibration process is performed, for example, when a new bulb 10 is inserted into the system, when the bulb 10 is transported, or optionally when a user defines a new chemistry for an existing system. Also, during these steps, the extinction coefficient e is kept constant, and k is given by the algorithm = (e'iF-'*e)-'*e'6F-1
Calculated according to

In Figure 4, two selected representative wavelengths 340n
A graphical vector representation of the absorbance scattering plot for m and 380 n+n is presented in the determination of the k value.

In reality, there are five wavelengths obtained from the 10 wavelength monochromator 204. If 5 wavelengths are used, 2
The diagram shown in the dimensional graph is effectively transformed into a five-dimensional diagram. The contour 100 shown in the graph is typical of correlated noise, and instead of being drawn only in two dimensions, it has five dimensions according to the number of wavelengths considered. A signal line 101 shown in the graph indicates the relative amount of absorbance change due to a change in the concentration of chromophore in the sample.

In practice, the noise is associated as represented by the oval representation 100 and the two absorbances 340 and 3
A scatter plot for these average values at 80 is shown. This scattering plot provides an F matrix of dispersion and covariance for these wavelengths.

The solution to get the best value i.e. length and direction is
It is determined by two mathematical expressions.

First, k = 1, where e is the extinction coefficient of the chromophore. In the second expression, ' is minimized for k large F6. F is the variance matrix of the noise, ie it defines the noise 100.

Axis 102 represents the length of the error distribution, and axis 103 represents the width of the error distribution.

The condition e=1 is represented in FIG. 4 by having all possible vectors with endpoints on the dashed line that are perpendicular to the extinction coefficient direction e. The next task is to calculate the mean squared error by 6F among all vectors that satisfy this condition.
The goal is to select the optimal k that minimizes '.

The technique used to find the extinction coefficient of a chromophore for a given chemistry at a particular set of wavelengths is Deming's method of finding the best linear fit to noisy data. in-
It is a film formation.

The dispersion matrix G is determined by observing the change in absorbance with respect to the average of absorbance in different bearings for a set of wavelengths. These observations are made while the chemical chromophore is changing within the cuvette.

The matrix G therefore reflects the relative amount by which the absorbance changes at these wavelengths, as well as the error characterized by F. The relative extinction coefficient is obtained from G without allowing skewing from F-type errors.

First, F undergoes eigenvector analysis. A unitary transformation U is found that rotates the system of absorbance coordinates with respect to these eigenvectors. Find a second transformation V whose matrix consists only of diagonal components and which corresponds to the diagonal component which is the square root of the eigenvalue. This, along with U, makes F the unit matrix F''=VUFU
It has the effect of converting 'V'=I. G is then transformed by the same transformation G"=VUGU'V' and also subjected to another eigenvector analysis. The eigenvalues of this latter matrix are denoted by F with large values of - for chemical changes. and all other values close to 1.

With the plurality of original data values that determined G transformed by VU, these dispersion matrices are just G. Deming's method is used to obtain the best fit line that minimizes the mean square orthogonal distance of these transformed points to the fit line. The components of e are then found by transforming this eigenvector back to the original absorbance coordinate system.

This method also allows checking the purity of the chromophore being measured. If two or more eigenvalues of G'' are substantially greater than -, this indicates the presence of other variable chromophores.

These extinction coefficient values are simply relative values since no concentration information was included.

7'-unit and the circuitry of the electronic unit for processing intensity signals at different wavelengths.

FIG. 5A shows the integrated circuit board or integrator circuit board and wavelength multiplexing support of the photometer assembly in block form. 5B and 5C depict a block diagram representation of a log 80G or log/analog-to-digital circuit board.

The functional and mechanical requirements for the integrator optical detector are as follows:
, an integrator circuit board 200 consisting of ten integrator circuits 202 , a combination logic control circuit 210 , and a calibration circuit 207 . The optical detector circuit measures the pulsed light input from monochromator 204 and converts the energy pulses into electrical signals proportional to the time integral of the current pulses.

Photodiode assembly 232 of detector assembly 201 is soldered to electronic assembly unit 26, which is mounted to monochromator assembly 27. Detector assembly 201 rides directly onto a 10-slit mask plate order sorting filter 25. Each slit 233 of the mask has a diameter of 340 nm (
It operates as a 5 nm bandpass filter tuned to a specific central wavelength within the optical spectrum from nanometers to 700 nm. Each photodiode sensor is aligned with a particular slit 233 in the mask.

Each photodiode is connected to a dense integrator circuit 202 for measuring the total light energy, ie, the time integral of the transmitted light pulse. All integrator circuits 202 are switched on in parallel and measure the single pulse energy at each wavelength.

The output signal from integrator circuit 202 is communicated to wavelength multiplexer assembly 205.

This multiplexer circuit 205 receives five signals 206 from ten integrator circuits for additional signal processing.
Gives the ability to choose.

The gain or sensitivity of the inch grater circuit is different for each wavelength and depends on the spectral responsivity of the detector, the spectral intensity curve of the xenon flashbulb 10, and the transmission of the monochromator. Capacitor values for the various circuits are selected to compensate for the total change in signal transmission. Each signal channel provides an output between 0 and 10 volts that is directly proportional to the time integral of the input current pulse. The integrator capacitor value determines the sensitivity of the circuit, and the M capacitor value or circuit sensitivity is based on the complete optical system, detector assembly 201 and flashbulb 10.

The energy from the bulb 10 converts the flash into a flash with an average signal level controlled by adjustment of the bulb supply voltage. The output signal at various wavelengths is -7,0 to -
It drops to within 10,0 volts. The flash of the light bulb is 12
It occurs at a nominal rating of 0 pps (7 second pulse) and the optical pulse width is approximately 1.5 usec. Each detector acts as a current source during light exposure and as an open circuit (high impedance) during dark intervals.

Programmable calibration circuit 207 provides three levels of signal current to measure integrator sensitivity and signal range. These signals are used for linear calibration during system linear testing and for diagnostic testing in both system and assembly testing.

Calibration circuit 207 consists of a regulated reference source 233, a precision divider network 234, and four multiplexers 208. The voltage source is connected to a divider network 23
2, with the output terminal connected to the three resistors in 4;
It comes from the 5 volt shunt regulator. The divider output is 0.0% of the total reference voltage. Old, 0.1. and 1. This is the ratio of Q.

The calibration signal is capacitively coupled to each integrator circuit 202, with each integrator circuit coupled via a numerically matched capacitor to the capacitor of the integrator used in each circuit. This results in an output voltage that is nominally equal to the step voltage generated by reference source 233.

The calibration signal is provided by a regulated +8.58 VDC VDC voltage source 233. Test point TPI is standard't
Provided to monitor M pressure. The calibration signal is 4
This occurs by switching the output path of one input multiplexer 208 from ground to one of three DC voltages. One of the four decoders internal to the multiplexer decodes the two line binary code to select the calibration stage programmed. Two externally held address lines are required to control the multiplexer. Address lines 230 and 231 come from connector P2 and are driven by the oven collector inverter. Multiplexer 208 is +15VD
It is a CMO5 device operating from a C source. The logic line terminates with a pull-up resistor at 4.7.

Each sensor within detector 201 provides a current input to a high speed precision integrator circuit. Circuit 202 is a high speed, low bias current (3pa max), low offset voltage amplifier, high stability low loss integrator capacitor and JFE
It consists of a T (junction field effect transistor)/analog switch discharge circuit.

A two-switch discharge circuit provides an integrator reset (RES) signal to reduce the total junction leakage current during the signal integration interval.
ET') circuit. During the integration time, both switches are turned off and 1. The J P E T switch is not biased and the analog switch is open. An output signal is applied through two switch networks between the amplifier output path and the general junction.

To reduce the total junction leakage current, a 750 ohm resistor is connected between the two switches and ground to provide a low impedance path from the analog switch to ground for sludge leakage current.

This reduces the JFET's drain to source voltage to a few millivolts, greatly reducing the total junction error current.

The JPET switch is turned off by switching the gate'H voltage to a 5VDC bias source. All analog switches are closed with a logical true (high) input signal. Matching complementary signals necessary to drive the circuit are provided at the transparent latch outputs "Q" and [Q★J. Two analog switches and one JFET switch are used in each integrator circuit. The above JFF, T switch and one analog switch are 1 Kariki integrator.
Used in the reset or discharge path of the capacitor. A second analog switch provides a negative bias to the gate junction of the JFET during the integration time. All circuits are switched in parallel and controlled by a single human power line INTEG.

In typical system operation, integrator circuit 2
02 is switched on, the indegrator offset is measured, the bulb flashes, and the signal level is measured and held.

Following an approximately 110 usec bulb flash, the signal signal is sampled and processed. Although the integrator circuit 202 is reset after sampling, the integrator normally resets approximately 3 milliseconds after the bulb flashes.

Capacitor values are selected to provide approximately equal signals at all wavelengths to the xenon flashbulb source 10, monochromator assembly 204, and a glass cuvette filled with deionized water.

The analog signal output from the integrator circuit 202 is
In order to supply multiple power to the wavelength multiplexer circuit 205, the multiplexer interface connector P2
sent to.

The J P E T switch injects charge error when the integrator 202 is switched on. A negative transition at the JFET gate junction is coupled to the integrator's total junction via the gate to train capacitance. This charging error results in a positive output offset error. The error voltage is inversely proportional to the integrator capacitor value.

Charge compensation is achieved by coupling a positive voltage step through a +2 pf capacitor to the overall junction of the integrator. During the integrator reset interval, the connected capacitor charges to the negative voltage determined by the regulation settings. Integrator 20
When 2 is switched on, the capacitor is switched to ground, transferring the charge of the capacitor to the total junction. The connected capacitor is maintained at ground during the integration interval and recharges during the reset. The compensation delay is made up of the single transistor switch delay time and the analog switch propagation delay time.

Each integrator circuit 202 requires two matching complementary logic signals to enable and reset the circuit. All integrators are switched in parallel and driven from the rQJ and rQ large J outputs of a single quad "D" latch. Multiple latch inputs are connected in common and driven by an oven collector inverter on multiplexer board 211.

All logic circuit drivers are latch circuits, and all drivers are open collector outputs. Two circuits require interface with the CMO5 calibration element, and
One driver connects to the TTL circuit.

All integrator analog outputs and ten OPAMP outputs are connected to connector P2 for mating with multiplexer circuit 205. 5 analog 5
IGGND line 206 is routed to connector P3. a plurality of analog signals are sent to the multiplexer circuit;
Five of the signals are sent back to the integrator circuit board 200 and sent to connector P3.

Multiplexer Board 211 Wavelength signal multiplexer circuit 211 provides a means for addressing the five analog signals 206 obtained from preamplifier integrator circuit 202 or preamplifier calibration circuit. The wavelength analog signals are cross-coupled to five multiplexers 205 to provide programmable selection of various signal combinations determined by system requirements. A calibration signal is connected to each multiplexer 205 to provide a diagnostic test signal.

Board 211 connects to preamplifier integrator circuit 202 via an interface connector. All logical address and data lines connect directly to multiplexer 211 via connector P1. All logic translation and data bus latches are included on the board. Analog output signal is integrator circuit board 2
Connected directly to 00. The output signal is usually LOG/
Single gain difference amplifier 217 on ADC conversion boat 212
Connect to.

The orb collector driver provides an interface TTL power to the CMO5 circuit on the integrator circuit board 200. Five wavelength signals are addressed for each chemical reaction test, one from each of multiplexers 205. For most chemical reactions, certain preferred wavelengths are specified.

Each of the five multiplexers 205 requires a 3-bit binary address to select one of the eight input signals. A total of 15 address lines come out of the three DJ hex latches. The multiplexer circuit 205 consists of five analog multiplexer chips, each with one of eight internal decoders. to -1 OVDC.

Ten analog input signals from integrator circuit board 200 are communicated to five multiplexer chips 205. The six inputs of each multiplexer 205 are one
It is obtained from 0 human power signals 213. The two remaining human powers 215 and 216 are connected to a reference voltage for diagnostic testing. The interconnection of the analog inputs provides a means for monitoring the various combinations of signal information required for a particular chemical reaction test.

Analog signals for human power 214 exit from programmable calibration circuit 207 on integrator circuit board 200 . This signal is controlled by setting a calibrator multiplexer 208 located on the integrator circuit board. Multiplexer 208 is controlled by two data hit lines 230, 231. Input 215 is typically connected to a fixed 13 VDC reference source located on integrator circuit board 200 . The TTI for the CMOS interface is determined for the two control signals 230, 231. Oven collector hex inverter integrator circuit board 20 with interface circuits for all control signals
Give to 0.

14o ADC Baud: 212 The 1og/8 DC board 212 is configured to connect to standard TTL logic circuits. Analog power is configured for a unique input signal driven from the available amplifier output. An analog signal line is connected to a differential amplifier with input terminals reversed, and a disconnection line is connected to the signal reference ground.

Analog human power is set to a +10 volt to -110 volt signal. The input signal can exceed 10 volts since the amplifier output used is limited to about 13 volts operating from a 15 volt supply. The assembly is designed to monitor signals within the 10 volt range. The log circuit is sized to receive a maximum input of ++,51 rt. The propagation measurements are limited to 10 volts, which is the ADC full scale signal.

All control lines are connected to the programmable array logic chip. The input impedance for each line is an ITTL gate. No terminal resistor network is provided on the board. All data bus lines connect to a three-state bus driver and one TTL quad latch.

All logic is driven from the latch I10 source.

FIG. 5b shows a block diagram of circuit board 212. FIG. L
The og/^DC board 212 consists of five identical log-converted signal channels. Each channel has a manual difference amplifier 217, an auto zero circuit 218, and a sample/hold
Amplification ffj (sample/hold amplif
ier) 219 and a log converter circuit 220.

The output signal from the log converter circuit 220 is connected to a 16 input multiplexer 221 for analog to digital conversion 5c. Multiple signal grounds for each channel are separated from analog and logic power grounds. Signal ground and AC analog ground are commonly connected at a star ground point. The logic ground is separated from the signal and analog ground paths, but inserted in common at the - point.

The signal input to each channel is connected to a difference amplifier 217 designed as a single gain inverter with common mode rejection of analog power ground noise. The signal from the difference amplifier 217 is connected to a test jack for external test monitoring and to an ADC multiplexer 221 for diagnostic testing and bulb source evaluation. The amplifier output is connected directly to the autozero offset subtraction circuit 218 to correct the voltage for dark signal offset and amplifier offset.

The reference switch is the auto zero output or OC reference 'r.
A pressure is selected as the manual force for the sample/holding circuit 219.

A DC reference voltage is provided as a standby input to the log converter circuit 220, or the signal is used for diagnostic testing. The sample/holding circuit 219 is the log converter circuit 2
20, or the signal is used for diagnostic testing. Sample/hold circuit 219 provides a constant signal to the log circuit during log conversion and data read times. The sample/hold circuit is also connected to an ADO multiplexer 221 for diagnostic monitoring.

The log signal output is directly connected to DC multiplexer 221. Multiplexer output 220 has eight DC235
It is connected to a voltage follower 223 that drives the human power to.

The DC converter provides a latched 16-bit output in two's complement format.

The analog input to each channel is connected to a single gain difference amplifier 217, which is connected as an inverter circuit. A precision thin film resistor network of four matched 50 resistors provides the matched resistor pairs necessary for 50 db of common mode noise rejection.

The analog power is connected to a signal lead connected to the reverse input, and is also connected to a signal ground connected to the non-reverse input. The non-reverse input driver is referenced to log channel signal ground. The circuit is designed for a minimum 50dB common mode rejection of voltage differences between grounds.

The preamplifier wavelength signal is at negative extreme pressure. Log converter circuits require a positive input current. For this reason, a signal inverter is required. The diagnostic signals from the preamplifier are both positive and negative extreme pressures. To this end, inverter circuit 217 is designed for input signals ranging from +10 volts to -110 volts.

Inverter output to test jack for test monitoring
Connected to ADC multiplexer 221 for diagnosis and also to autozero manual power.

Autozero offset subtraction circuit 218 provides offset voltage subtraction due to the dark signal offset and the inverter amplifier 217 offset. Circuit 218 consists of a low hysteresis storage capacitor driven by inverter amplifier 218. This capacitor is switched to ground during the offset charging time and switched to the reference switch during offset subtraction. When switched to ground, the capacitor charges to a voltage equal to the human input signal, typically 15+mV or less. The capacitor charges to a voltage equal to the human input signal plus the offset of the amplifier. The capacitor is grounded for a minimum of 600 usec to allow the capacitor to charge to the full offset voltage. The capacitor is then switched "in series" with the output of inverter 218 to subtract the offset error from the total human power signal.

Auto-zero charging time is software controlled,
Also, 600 usec or 62
．． Changes up to 0 ms. 10 between switching off the autozero charging cycle and applying the human power signal.
A delay in usec is allowed.

Autozero switching is controlled by both ZRO and LOGREF lines. When LOGREF is switched low, capacitor switching is controlled by the ZRO line, with ZRO high to ground and ZRO low (0).
is connected to the “difference” state. When LOGREF is switched high, AZRO is forced high and the capacitor is switched to ground. The ground path of the capacitor is completed through one analog switch circuit of reference switch 222.

The sample and hold input signals are determined by the setting of reference switch 222. One of the two signal sources is connected to sample/hold circuit 219. Reference switch 2
22 connects one of the reference voltage and autocello output to the sample/
Two single pole Nislow switches (throw 5wltch) connected to feed the hold circuit 219.

One switch is controlled by the LOGREF logic line and the second switch is controlled by AZRO. The reference voltage is set to provide a log input terminal slightly greater than the log reference current. This results in a small negative voltage from the log circuit.

Sample and holding circuit 219 for each log converter 220
provides a constant input terminal during relatively long conversion and data read times. In the "sample" state, the output signal tracks the input and also tracks changes in the human input signal. The human power signal seen by sample/hold circuit 219 is the difference between the total human power signal and the voltage at the autozero capacitor.

Low level signal measurements and long log conversion times require sample/hold circuit 219 to be a low drift circuit. The gain of the log circuit is high for low level signals and therefore the drift is amplified during long data conversion intervals. Larger value capacitors reduce drift to the required rate, but increase acquisition time. The minimum sample time to allow acquisition to within 0.1 of the pre-signal is 5
．． Ousec.

During the long data wait interval, the sample/hold circuit is switched to the "sample" state and the 1, OCRεF signal is switched on.

This is the log conversion circuit 220 that prevents saturation of the log circuit.
A fixed standby current is supplied to the

The sample/hold circuit 219 is switched in parallel and controlled by the SMP L line. SMPL is low to switch all circuits to the "sample" state and all circuits of the switched high set to the "hold" state.

Offset adjustments are made to each sample/hold circuit 219 to correct for log converter input offset voltage errors. The offset a adjustment provides a means for adding an error voltage to compensate the log circuit. Linear adjustments are made with precise step voltages.

A log converter circuit 220 provides an analog output voltage proportional to the logarithm of the input current. Log converter 220 requires a set of reference currents to define the energy current to zero output volts. The log converter circuit 220 consists of 10
The log chip is set to 7 o 62 holts per input terminal of , and the log chip is designed to divert the positive input i. The circuit is designed for voltage input. The output signal is buffered using a buffer amplifier built into the chip. The output is directly connected to a DC multiplexer 221 for digital processing.

Multiplexer 221 is a 16 channel device that provides a means for continuously or optionally monitoring one of 16 input terminals from various signals or diagnostic points. The multiplexer output 222 is connected to a high power impedance voltage follower 223 to reduce input signal source loading. The output impedance of follower 223 is low and A
Preventing DC measurement errors. The follower drives an ADC 235 with an input impedance of 10K.

Multiplexer 221 requires four lines of binary code obtained from external data latches. Inside multiplexer 221 is a decoder for selecting one of the 16 channels.

A delay time of 25 usec is allowed for multiplexing and signal temporal processing time. This means that data reading takes 25 uses after the multiplexer clock pulse.
c means to be late. The power of multiplexer 221 can address any arrangement required for a particular application. During normal system operation,
Absorbance readings are determined by monitoring only the log signal. All signals can be monitored during diagnostic tests.

Multiplexer 221 manpower falls into two basic categories. a log-converted signal for absorbance measurements;
The diagnostic signal and the first five inputs are log converter signals, and the next five inputs are the propagation signal and/or the preamplifier diagnostic signal. The third group of 5 signals are diagnostic signals obtained from the 21 sample/hold outputs. The final human power source is a 2.5 volt reference voltage source.

The ADC converter 235 is a 16-bit successive approximation converter with a maximum conversion time of 35 usec. A
The input impedance of the ADG 235 is
It is strapped to a K ohm. The human power pin has three signals f1224: (1) analog ground, (2)
◆2.5vdc standard and (3) multiplexer 221
can be jumpered to one of the outputs of.

The analog ground connection is jumpered to the input for zeroing. The 2.5 volt reference is connected to a pin that is jumpered to DC power for diagnostic testing. The ADC power is usually a multiplexer 221
is jumpered to the output of

The ADO output is latched 16-bit data in two's complement format. The previous data remains latched until the next data conversion pulse. A status line is provided to indicate that the DC 235 is in a conversion cycle.

The end of conversion is determined by monitoring the status signal and detecting a trailing edge change. Inverter, chip 22
One gate of drives the output signal line. The “end of conversion” pulse (busy) is a negative going signal (goi
ng signal) and the end of the transformation is indicated by a positive change in the trailing edge.

The 16 data lines are connected to two 8-bit, three-state buffers. Data is read in two bytes, each byte being read as fast as required by the system program.

The devices, systems and methods of the present invention allow concentration data of analytes in a sample to be determined more accurately and with greater efficiency than previously possible. A suitable program is implemented electronically based on the instructions provided to enable the necessary data determination.

The output from the electronic unit as absorbance values in digital form is presented as raw absorbance values to a central processing unit running under a program that causes the linear combination to be calculated. Constants e, f and k are determined and the bait record absorbance values are developed into estimated absorbance values. The next step is to use a polychromatic matrix, using e and k to determine said matrix, to develop a special evaluation absorbance set. Estimated concentrations using multiple constants k are obtained by the linear combination. An indication of the presence of buffer substances is also obtained.

A second central processing unit operates on the density representation to smooth and interpolate the data and to develop a regression line in a particular window of data, thereby determining the average value of the data in the window and the average value of the data in the window. Get information like slope.

Although specific examples are expressed, it is understood that many variations differ from each other only in detail. For example, although linear coupling is described for five wavelengths, the same principles can be applied to smaller or larger sets of wavelengths. This may be, for example, 2.3 or 4.6.7 or 8, or multiple sets of several hundred wavelengths. For example, for a system producing 128 wavelengths, 10 wavelengths can be selected for linear combination. Also, although the expressions relate to the concentration of a single chromophore substance, the concentration of more than one chromophore substance in a given sample can be determined.

[Brief explanation of the drawing]

Fig. 1 is an optical diagram of a monochromator according to the present invention, Fig. 2 shows the sections of the parts of the photometer seen from an isometric angle, and Fig. 3 shows the housing of the monochromator together with the stripped parts. 4 is an absorbance scattering plot, a graph illustrating the relationship of signal and noise at these two selected wavelengths to obtain an optimal linear combination of absorbance values at these two selected wavelengths; FIG. Figure 5A, 5th
Positional relationship indication diagram of Figure B and Figure 5C, Figure 5A, Figure 5
Figures B and 5C are block diagrams illustrating the electronic unit coupled to the detector of the photometer. 10: Flash light bulb! 1, 13, 17, 120: Ray, 15: Lens, 18.21: First and second collimator mirror 19:
Parallel rays, 22: Convergent rays, 24: Detector array, 25: Order classification filter, 26: Electronic unit.

Claims (61)

[Claims] (1) A method of measuring the concentration of at least one chromophore substance in a sample, comprising: a) flashing a light bulb to cause a flash of light to pass through the sample containing the chromophore substance; b) A method for measuring concentration, comprising: measuring absorbance values with said sample at different wavelengths; c) determining a linear combination of said absorbance values at said different wavelengths, thereby obtaining a concentration value of said chromophore. 2. The method of claim 1, wherein errors in absorbance values are correlated to each other at different wavelengths, and the linear combination is determined based on this correlation. (3) the linear combination reflects the nature of the correlated errors and the direction of the extinction coefficient of the chromophore in the sample;
It also includes measuring a change in the concentration of the chromophore.
The method according to claim 2. 4. The method of claim 3, comprising: (4) measuring the absorbance values for five different wavelengths. (5) a) where a is a column matrix of absorbance values, c is a chromophore concentration in a scalar quantity, e is a column matrix of extinction coefficients of the chromophore, and f is a column matrix showing errors, a=
b) Determining said concentration by applying Beer's law formulated as c*e+f, b) expressed as k*F*k' if the constraint k*e=1 and the mean squared error is minimum. determining the linear combination according to noise, where k is a row matrix of coefficients of the linear combination, F is a covariance error matrix of f, and k' is a transposed matrix of k; c ) e' is the transposed matrix of e and F^-^1 is the inverse matrix of F, the formula k = (e'*F^-^1*e)^-^1*e'*F^-
Claim 3 comprising determining the elements of k according to ^1.
The method described in. 6. The method of claim 5, wherein the error matrix F is determined by measuring changes in the level of absorbance from a flash of light passing through a certain medium. 7. The method of claim 6, wherein the error matrix F is measured for different combinations of pairs of wavelengths. (8) The method of claim 7, wherein the different combinations are obtained from multiple sets of five wavelengths. (9) The extinction coefficient e is determined for the chromophore of the selected chemical reaction using a generalization of Deming's method and an eigenvector transformation to obtain the best linear fit to the absorbance value. The method described in. (10) Determination of said extinction coefficient comprises: a) a change in absorbance level determined during a change in the concentration of a chromophore of a chemical reaction of a sample flashed by said light bulb, said absorbance level for a plurality of pairs of wavelengths; Determine the dispersion matrix G by observing the change in the absorbance level with respect to the average of the changes, from which the matrix F
said matrix G reflects the relative amount by which absorbance levels change at pairs of wavelengths in addition to said error characterized by; and b) removing data representing errors in said matrix;
10. The method of claim 9, thereby comprising obtaining a relative extinction coefficient from the matrix G. (11) subjecting said matrix F to an eigenvector analysis, obtaining a unitary transformation for converting coordinates to eigenvector coordinates, thereby transferring absorbance levels to eigenvector coordinates; a second transformation of said transformation; obtaining a second transformation in which a matrix includes only diagonal elements, said diagonal elements being the square roots of said eigenvectors;
The method according to claim 10. (12) The dispersion matrix G is transformed by a unitary transformation and a second transformation, determining the values of said eigenvectors and the transformed dispersion matrix G, and calculating the mean square orthogonal distance of the transformed points to the line according to Deming's method. 11. The method of claim 10, comprising obtaining a minimizing best fit line, transforming the eigenvectors back to the original absorbance coordinate system, and obtaining components of the extinction coefficient e. 13. The method of claim 1, comprising: determining the change in concentration. 14. The method of claim 9, comprising: determining the change in concentration. (15) A single-beam system for determining the concentration of at least one chromophore in a sample, comprising: a light bulb for instantaneously emitting a beam of light through the sample; and dispersing the beam of light passing through the sample into multiple wavelengths. means for obtaining the intensity of said light beam signal at a plurality of selected wavelengths; means for determining absorbance values from said intensity values at different wavelengths; and means for determining absorbance values at two or more wavelengths. means for selecting a linear combination of , thereby obtaining a concentration value of said chromophore. (16) a monochromator for splitting the light beam from the flash bulb and a lens for splitting the light beam from the flash bulb, the monochromator directing the light beam from the diverging light beam to a diffraction grating; a first collimator mirror for producing parallel light rays, the diffraction grating of which separates the parallel light rays according to the wavelength; a second collimating mirror for converging the light beam at a plurality of selected positions of the converging light beam, the second collimating mirror having the surface disposed in a preselected plane for different wavelengths of the converging light beam; 16. The system of claim 15. (17) The system according to claim 16, further comprising an order classification filter in the vicinity of the preselected surface. (18) a toroid mirror for directing the light beam from the flashbulb through the sample holder; and a slit, wherein a selected portion of the light beam passes through the slit and is directed toward the first collimator mirror. 2. A slit disposed close to the lens as shown in claim 1.
The system described in 7. (19) a detector array in the preselected plane that receives light rays of at least 10 different wavelengths; and a detector array disposed in front of the detector array such that the selected light rays of different wavelengths pass through the detector array. a row of slits,
The system of claim 18. (20) an electronic unit connected to said detector array for receiving an intensity signal of a selected wavelength of said light beam in analog form; means for processing; processor means for processing said digital data to obtain a linear combination of absorbance values at said different wavelengths; and processor means for determining successive concentration values from said absorbance values. 20. The system of claim 18, comprising: (21) comprising means for determining the change in concentration;
16. The system of claim 15. 22. The system of claim 20, comprising means for determining concentration changes. (23) A method for determining the extinction coefficient for a chromophore of a selected chemical reaction according to Deming's method and eigenvector transformation in order to obtain the best linear fit to the variation of the absorbance value in the chemical reaction, the method comprising: a change in absorbance level determined during a change in the concentration of a chromophore of a chemical reaction in a flashing sample, by observing the change in absorbance level for an average of changes in average absorbance level with respect to multiple pairs of wavelengths; A method for determining an extinction coefficient, wherein a dispersion matrix G is determined, whereby the dispersion matrix reflects the relative amount by which absorbance levels vary at selected pairs of wavelengths. (24) dispersion matrix F further includes an error characterized by a dispersion matrix of error matrices, and removes data representing said error matrix, thereby obtaining the relative extinction coefficient of said chromophore from said dispersion matrix; 24. The method of claim 23, comprising: (25) subjecting said error matrix to eigenvector analysis, obtaining a unitary transformation, thereby transferring absorbance coordinates to eigenvector coordinates; and a second transformation, the matrix of said transformation consisting only of diagonal elements; 25. The method of claim 24, comprising obtaining a second transformation where the diagonal elements are square roots of the eigenvectors. (26) The variance matrix G is transformed by a unitary transformation and a second transformation, obtaining the best common line that minimizes the mean square orthogonal distance to the line according to Deming's method of the transformed points, 26. The method of claim 25, comprising converting back to the original absorbance coordinate system and obtaining the extinction coefficient. (27) A method for measuring the concentration of a chromophore substance in a sample, in which a is a column matrix of measured absorbance values, c is the chromophore concentration in a scalar quantity, and e is the chromophore concentration of a selected chemical reaction. determining said concentration by applying Beer's law formulated as a column matrix of extinction coefficients and a=c*e+f, where f is a column matrix indicating the error;
Constraint k*e=1 and k if the mean squared error is minimum
The linear combination according to the noise represented by *F*k', where k is a row matrix of the coefficients of the linear combination, F is the covariance error matrix of f, and k' is the transposed matrix of k. , the formula k=(e'*F^-^1*e)^-^1*e, where e' is the transposed matrix of e and F^-^1 is the inverse matrix of F.
A concentration measurement method comprising determining an element of k according to '*F^-^1. 28. The method of claim 26, wherein the error matrix F is determined by measuring changes in the level of absorbance from a flash of light passing through a medium. (29) The method of claim 28, wherein the error matrix F is measured for different combinations of pairs of wavelengths. 30. The method of claim 29, wherein the different combinations are obtained from multiple sets of five wavelengths. (31) A method for determining the linear combination of absorbance values at different wavelengths of light rays passing through a chromophoric substance in a sample, the method comprising: a) producing a flash ray passing through said sample containing said chromophore substance; flashing a light bulb; b) measuring the absorbance values with the samples at different wavelengths, wherein the errors in the absorbance values are correlated at different wavelengths, and the linear combination uses this correlation; A linear combination determination method, including: (32) The linear combination reflects the nature of correlated errors and the direction of extinction coefficients of chromophores in the sample, and also includes measuring changes in the concentration of the chromophores. The method according to item 31. 33. The method of claim 32, comprising measuring the absorbance values for five different wavelengths. (34) A method for determining the concentration of at least one chromophore substance in a sample in a single beam system, the method comprising: instantaneously generating a beam of light through the sample; obtaining signal intensities of said light beam at a number of selected wavelengths;
determining absorbance values from said intensity values at different wavelengths;
A method for determining concentration, comprising selecting a linear combination of absorbance values at two or more wavelengths, thereby obtaining a concentration value for said chromophore. (35) branching the light beam from the flash bulb; directing the branched light beam from the branched light beam to a first collimator mirror for producing parallel light beams; and a diffraction grating that divides the parallel light beam into a number of light beams according to wavelength. directing the parallel light beam toward a second collimating mirror and reflecting a plurality of branched light beams into convergent light beams directed at a plurality of selected positions on a surface;
3. Processing the intensity of the light beam at the surface at different wavelengths to determine absorbance as a concentration value.
The method described in 4. (36) operatively connecting a detector array in said plane, thereby obtaining an intensity signal of a selected wavelength of said light beam;
36. The method of claim 35, wherein the intensity signal is processed to obtain absorbance values at different wavelengths. (37) The method of claim 36, wherein the change in concentration is determined. (38) A system for determining the concentration of at least one chromophoric substance in a sample, comprising: a) a light bulb for instantaneously generating a light beam through the sample containing the chromophore substance; and b) a different c) means for determining a linear combination of the absorbance values at the different wavelengths, thereby obtaining a concentration value of the chromophore; , concentration determination system. 39. The system of claim 38, wherein errors in absorbance values are correlated to each other at different wavelengths, and further comprising means for determining the linear combination reflecting this correlation. (40) The linear combination reflects the nature of the correlated errors and the direction of the extinction coefficient of the chromophore in the sample and also includes means for measuring changes in the concentration of the chromophore. 40. The system of claim 39. 41. The system of claim 40, comprising means for measuring the absorbance values for five different wavelengths. (42) The method of claim 1, wherein an optimal linear combination is determined. (43) The method of claim 31, wherein an optimal linear combination is determined. (44) comprising means for determining an optimal linear combination;
39. The system of claim 38. (45) The optimal linear combination is defined by the constraints k*e=1 and k
*F*k' is determined according to the minimum, where k reflects the row matrix forming the coefficients of said linear combination;
e is a column matrix of extinction coefficients of chromophores, and F
is a covariance error matrix, and the elements of k are expressed by the formula k=(e'*F^-^1*e)^-^1*e'*F^-
43. The method of claim 42, where e' is the transposed matrix of e and F^-^1 is the inverse matrix of F. (46) The method according to claim 42, wherein the number of wavelengths is 3 or more. (47) A set of coefficients for linear coupling for use in measuring absorbance values of a sample using the flashphotometer with correlated noise regarding the flash of the flashphotometer, with the constraint k* has e=1 and a minimum k*F*k',
where k is the row matrix forming the coefficients of said linear combination, e is the column matrix of the extinction coefficients of the chromophores in the sample of the substance whose absorbance is to be determined, and F is the covariance error matrix; Furthermore, the elements of k are expressed as k=(e'*F^-^1*e)^-^1*e'*F^-
^1, where e' is the transposed matrix of e and F^-^1 is the inverse matrix of F, the coefficients for the linear combination. (48) A single-beam system for determining the concentration of at least one chromophore substance in a sample, comprising: a light bulb for instantaneously emitting a beam of light through the sample; and a light bulb for instantaneously emitting a beam of light through the sample; A single beam system comprising means for spectroscopy and means for obtaining signal intensities of said beam at a selected number of wavelengths. (49) comprising a monochromator for splitting the light beam from the flashbulb and an optical element for directing the light beam from the flashbulb, the monochromator directing the light beam from the diverging beam to a diffraction grating. a first collimator mirror for producing parallel light rays, the diffraction grating of which separates the parallel light rays according to the wavelength; a second collimating mirror for converging the light beam at a plurality of selected positions of the converging light beam, the second collimating mirror having the surface disposed in a preselected plane for different wavelengths of the converging light beam; 50. The system of claim 48. (50) The system of claim 49, including an order classification filter disposed with respect to the surface. (51) directing the light beam from the flashbulb through a sample holder through a slit, the light beam being positioned proximate to the lens such that a selected portion of the light beam passes through the slit and is directed toward the first collimating mirror; 51. The system of claim 50, including a toroidal mirror for directing to the slit. (52) a detector array on the surface that receives light rays of at least 10 different wavelengths; and a slit array disposed in front of the detector array such that the selected light rays of different wavelengths pass through the detector array. 52. The system of claim 51, comprising: (53) an electronic unit connected to said detector array for receiving an intensity signal of a selected wavelength of said light beam in analog form, and said analog for obtaining digital absorbance, such as data related to said intensity signal; means for processing an intensity signal; processor means for processing said digital data to obtain a linear combination of absorbance values at said different wavelengths; and a processor for determining successive concentration values from said absorbance values. 52. The system of claim 51, comprising means. (54) The system of claim 53, determining the change in concentration. (55) A device operatively connected to an output detector array of a monochromator from a single beam arrangement, the output detector array providing intensity signals of selected wavelengths of the light beam for the sample in the path of the beam. in analog form and comprising means for processing said analog intensity signal to obtain digital absorbance signal data. (56) Processor means for processing the digital data to obtain a linear combination of absorbance values at the different wavelengths, and processor means for determining successive concentration values from the absorbance values. 55. The device according to 55. (57) The apparatus of claim 56, comprising means for determining concentration changes. (58) said analog intensity signals are at ten wavelengths, and multiplexer means for receiving said signals, means for outputting intensity signals of five wavelengths, and said five wavelength intensity signals; 56. The apparatus of claim 55, comprising means for converting into log-digital signals representing absorbance values at the five wavelengths. 59. The apparatus of claim 58, including means for multiplexing an absorbance value analog signal into an absorbance digital signal to provide output to the processing means. (60) The processor means process the linear combination according to a constraint K*e=1 and a noise represented by K*F*K' when the mean squared error is minimum, where K is the linear combination is a row matrix forming the coupling coefficients, F is f
and K′ is the transpose matrix of K, e is a column matrix of extinction coefficients of the chromophore, and f is a column matrix representing the error.
60. Apparatus according to claim 59. (61) The apparatus of claim 60, wherein the linear combination is an optimal linear combination.